Coryneform bacteria are industrial microorganisms that produce chemical substances having various applications in the industry of animal feed, drugs, food, and the like, which contain L-lysine, L-threonine and various nucleic acids. To develop high titer strains from such coryneform bacteria by genetic engineering or metabolic engineering, the expression of genes involving several metabolic pathways in coryneform bacteria should be selectively regulated. Thus, promoter sequences useful for this gene regulation are required.
Upon the gene expression in coryneform bacteria, genes are generally expressed from their own promoters (Vasicova, P., et al., J. Bacteriol. 181, 6188-6191, (1999), etc.). However, unlike other industrial microorganisms such as Escherichia coli and Bacillus subtilis, there is no information on the basic structure of promoter sequences for gene expression in coryneform bacteria. For this reason, promoters have been developed by eliminating promoter regions from genes associated with resistance to antibiotics such as chloramphenicol, introducing chromosomal DNA isolated from coryneform bacteria and digested with suitable restriction enzymes into the promoter sites, transforming coryneform bacteria with the resulting DNA molecules, assessing antibiotic resistance of obtained strains (Eikmanns, B. J., et al., Gene, 102, 93-98, (1991); Patek, M., et al., Microbiology, 142, 1297-1309, (1996)). However, previously developed promoter sequences still need to be improved with respect to selective expression and expression efficiency of genes of interest.
Conventional methods involving promoter isolation include (1) the use of a promoter probe vector system in the random cloning of genomic DNA fragments upstream of a reporter gene expressed only when a cloned fragment contains promoter activity; (2) gene-specific probe-based hybridization by which a gene and its promoter are isolated from a genomic library; and (3) differential hybridization of an inducible cDNA probe and a non-inducible cDNA probe to a gene bank.
Recently developed comparative proteome analysis by two-dimensional (2-D) gel electrophoresis is a technology that identifies proteins differentially expressed at different physiological states. Based on differentially expressed proteins, many studies have been performed to identify regulatory genes capable of increasing expression of the proteins.
Based on this background, the present inventors conducted 2-D gel electrophoresis of proteins whose expression was induced in the presence of lysine, and detected and identified proteins displaying differential expression patterns by comparative analysis of proteomes. When polynucleotides corresponding to the putative promoter regions of the identified proteins were amplified by PCR and were introduced upstream of a promoter-deficient lacZ gene, a remarkable increase in beta-galactosidase activity was observed in the presence of lysine, thereby leading to the present invention, which provides novel promoter nucleic acid molecules capable of inducing gene expression in the presence of lysine.